Differentially methylated genes involved in reproduction and ploidy levels in recent diploidized and tetraploidized Eragrostis curvula genotypes.

Epigenetics studies changes in gene activity without changes in the DNA sequence. Methylation is an epigenetic mechanism important in many pathways, such as biotic and abiotic stresses, cell division, and reproduction. Eragrostis curvula is a grass species reproducing by apomixis, a clonal reproduction by seeds. This work employed the MCSeEd technique to identify deferentially methylated positions, regions, and genes in the CG, CHG, and CHH contexts in E. curvula genotypes with similar genomic backgrounds but with different reproductive modes and ploidy levels. In this way, we focused the analysis on the cvs. Tanganyika INTA (4x, apomictic), Victoria (2x, sexual), and Bahiense (4x, apomictic). Victoria was obtained from the diploidization of Tanganyika INTA, while Bahiense was produced from the tetraploidization of Victoria. This study showed that polyploid/apomictic genotypes had more differentially methylated positions and regions than the diploid sexual ones. Interestingly, it was possible to observe fewer differentially methylated positions and regions in CG than in the other contexts, meaning CG methylation is conserved across the genotypes regardless of the ploidy level and reproductive mode. In the comparisons between sexual and apomictic genotypes, we identified differentially methylated genes involved in the reproductive pathways, specifically in meiosis, cell division, and fertilization. Another interesting observation was that several differentially methylated genes between the diploid and the original tetraploid genotype recovered their methylation status after tetraploidization, suggesting that methylation is an important mechanism involved in reproduction and ploidy changes.

An exonuclease V homologue is expressed predominantly during early megasporogenesis in apomictic Brachiaria brizantha.

MAIN CONCLUSION: An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

Identification of IPT9 in Brachiaria brizantha (syn. Urochloa brizantha) and expression analyses during ovule development in sexual and apomictic plants.

BACKGROUND: In Brachiaria sexual reproduction, during ovule development, a nucellar cell differentiates into a megaspore mother cell (MMC) that, through meiosis and mitosis, gives rise to a reduced embryo sac. In aposporic apomictic Brachiaria, next to the MMC, other nucellar cells differentiate into aposporic initials that enter mitosis directly forming an unreduced embryo sac. The IPT (isopentenyltransferase) family comprises key genes in the cytokinin (CK) pathway which are expressed in Arabidopsis during ovule development. BbrizIPT9, a B. brizantha (syn. Urochloa brizantha) IPT9 gene, highly similar to genes of other Poaceae plants, also shows similarity with Arabidopsis IPT9, AtIPT9. In this work, we aimed to investigate association of BbrizIPT9 with ovule development in sexual and apomictic plants. METHODS AND RESULTS: RT-qPCR showed higher BbrizIPT9 expression in the ovaries of sexual than in the apomictic B. brizantha. Results of in-situ hybridization showed strong signal of BbrizIPT9 in the MMC of both plants, at the onset of megasporogenesis. By analyzing AtIPT9 knockdown mutants, we verified enlarged nucellar cell, next to the MMC, in a percentage significantly higher than in the wild type, suggesting that knockout of AtIPT9 gene triggered the differentiation of extra MMC-like cells. CONCLUSIONS: Our results indicate that AtIPT9 might be involved in the proper differentiation of a single MMC during ovule development. The expression of a BbrizIPT9, localized in male and female sporocytes, and lower in apomicts than in sexuals, and effect of IPT9 knockout in Arabidopsis, suggest involvement of IPT9 in early ovule development.

Reproductive and Agronomic Characterization of Novel Apomictic Hybrids of Paspalum (Poaceae).

The tetraploid germplasm of Paspalum contains a large diversity that can be used to generate better forages. The objective was to evaluate a group of Paspalum notatum and Paspalum simplex apomictic hybrids for a set of agronomic traits and apomixis expressivity. Forage yield, cold tolerance, winter regrowth, and seed yield were evaluated. The expressivity of apomixis was evaluated in P. simplex hybrids by flow cytometry. Progeny testing with molecular markers was used to determine the genotypic variability in the progeny. Differences within P. notatum and P. simplex hybrids were observed for all traits, and some of them were superior in comparison with the controls. The accumulated forage yield during three years was 988 g m-2 in the P. notatum hybrids, whereas, in P. simplex, the average forage yield per harvest (40 days of regrowth) was 180 g m-2. In P. simplex, the apomixis expressivity varied between 0 and 100%, and 65% of the hybrids showed high apomixis expressivity (superior to 70%). The genotypic mean homogeneity in the progeny was 76% and 85% in P. notatum and P. simplex, respectively. The generation of hybrids with high apomixis expressivity that combine good agronomic performance and homogeneity in the offspring is possible in tetraploid P. notatum and P. simplex.

From tetraploid to diploid, a pangenomic approach to identify genes lost during synthetic diploidization of Eragrostis curvula.

Introduction: In Eragrostis curvula, commonly known as weeping lovegrass, a synthetic diploidization event of the facultative apomictic tetraploid Tanganyika INTA cv. originated from the sexual diploid Victoria cv. Apomixis is an asexual reproduction by seeds in which the progeny is genetically identical to the maternal plant. Methods: To assess the genomic changes related to ploidy and to the reproductive mode occurring during diploidization, a mapping approach was followed to obtain the first E. curvula pangenome assembly. In this way, gDNA of Tanganyika INTA was extracted and sequenced in 2x250 Illumina pair-end reads and mapped against the Victoria genome assembly. The unmapped reads were used for variant calling, while the mapped reads were assembled using Masurca software. Results: The length of the assembly was 28,982,419 bp distributed in 18,032 contigs, and the variable genes annotated in these contigs rendered 3,952 gene models. Functional annotation of the genes showed that the reproductive pathway was differentially enriched. PCR amplification in gDNA and cDNA of Tanganyika INTA and Victoria was conducted to validate the presence/absence variation in five genes related to reproduction and ploidy. The polyploid nature of the Tanganyika INTA genome was also evaluated through the variant calling analysis showing the single nucleotide polymorphism (SNP) coverage and allele frequency distribution with a segmental allotetraploid pairing behavior. Discussion: The results presented here suggest that the genes were lost in Tanganyika INTA during the diploidization process that was conducted to suppress the apomictic pathway, affecting severely the fertility of Victoria cv.

A combined transcriptome - miRNAome approach revealed that a kinesin gene is differentially targeted by a novel miRNA in an apomictic genotype of Eragrostis curvula.

Weeping lovegrass (Eragrostis curvula [Shrad.] Nees) is a perennial grass typically established in semi-arid regions, with good adaptability to dry conditions and sandy soils. This polymorphic complex includes both sexual and apomictic cytotypes, with different ploidy levels (2x-8x). Diploids are known to be sexual, while most polyploids are facultative apomicts, and full apomicts have also been reported. Plant breeding studies throughout the years have focused on achieving the introgression of apomixis into species of agricultural relevance, but, given the complexity of the trait, a deeper understanding of the molecular basis of regulatory mechanisms of apomixis is still required. Apomixis is thought to be associated with silencing or disruption of the sexual pathway, and studies have shown it is influenced by epigenetic mechanisms. In a previous study, we explored the role of miRNA-mRNA interactions using two contrasting E. curvula phenotypes. Here, the sexual OTA-S, the facultative Don Walter and the obligate apomictic Tanganyika cDNA and sRNA libraries were inquired, searching for miRNA discovery and miRNA expression regulation of genes related to the reproductive mode. This allowed for the characterization of seven miRNAs and the validation of their miRNA-target interactions. Interestingly, a kinesin gene was found to be repressed in the apomictic cultivar Tanganyika, targeted by a novel miRNA that was found to be overexpressed in this genotype, suggestive of an involvement in the reproductive mode expression. Our work provided additional evidence of the contribution of the epigenetic regulation of the apomictic pathway.

Spotting the Targets of the Apospory Controller TGS1 in Paspalum notatum.

Sexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis.

Isolation of Boechera stricta Developing Embryos for Hi-C.

The possibility of analyzing chromatin topology in developing plant embryos is hampered by inaccessibility of the embryo sac, deeply embedded in the maternal seed tissue, following double fertilization. Here we describe a protocol to isolate, purify, and prepare developing Boechera stricta embryos for chromosome conformation capture-based methods as in situ Hi-C experiments. Early globular embryos can be isolated by air-pressure microaspiration, and subsequently washed to eliminate residual cells from the endosperm and maternal seed coat, allowing for pure sampling of selected stages of embryogenesis. This protocol allows for the possibility of comparing genome topology during plant embryonic differentiation since early until late embryo development stages.

Variation of Residual Sexuality Rates along Reproductive Development in Apomictic Tetraploids of Paspalum.

Most apomictic plants are facultative, maintaining the ability to reproduce sexually at different frequencies depending on the taxa, ploidy, and reproductive stage. In this context, Paspalum species are good model systems for studies evaluating the varying levels of apomixis expression. We aimed to identify, in apomictic tetraploid Paspalum species, the degree of apomixis and residual sexuality in three stages of reproductive development, and if their expression varies along them in order to predict their realized impact on the genetic diversity of future generations. Three main stages in the reproductive development (i.e., ovule, seed, and progeny) were studied in tetraploids from populations of P. cromyorhizon and P. maculosum. Mature ovules were studied using cytoembryological analysis, seeds by flow cytometry, and progeny tests with molecular markers. The expression of sexuality and apomixis was compared in each stage. We observed a decline in expression of sexual reproduction through the consecutive stages, jointly with an increase of apomixis expression. Both species showed at least one tetraploid plant capable of producing progeny by sexual means. These small rates of sexually originated progeny prove the ability of apomictic plants to produce low levels of genetic variation through rare events of sexuality. This study also demonstrates the importance of analyzing different reproductive stages in order to get a whole picture of the reproductive outcomes in plant evolution.

The Auxin-Response Repressor IAA30 Is Down-Regulated in Reproductive Tissues of Apomictic Paspalum notatum.

The capacity for apomixis in Paspalum notatum is controlled by a single-dominant genomic region, which shows strong synteny to a portion of rice chromosome 12 long arm. The locus LOC_Os12g40890, encoding the Auxin/Indole-3-Acetic Acid (Aux/IAA) family member OsIAA30, is located in this rice genomic segment. The objectives of this work were to identify transcripts coding for Aux/IAA proteins expressed in reproductive tissues of P. notatum, detect the OsIAA30 putative ortholog and analyze its temporal and spatial expression pattern in reproductive organs of sexual and apomictic plants. Thirty-three transcripts coding for AUX/IAA proteins were identified. Predicted protein alignment and phylogenetic analysis detected a highly similar sequence to OsIAA30 (named as PnIAA30) present in both sexual and apomictic samples. The expression assays of PnIAA30 showed a significant down-regulation in apomictic spikelets compared to sexual ones at the stages of anthesis and post-anthesis, representation levels negatively correlated with apospory expressivity and different localizations in sexual and apomictic ovules. Several PnIAA30 predicted interactors also appeared differentially regulated in the sexual and apomictic floral transcriptomes. Our results showed that an auxin-response repressor similar to OsIAA30 is down-regulated in apomictic spikelets of P. notatum and suggests a contrasting regulation of auxin signaling during sexual and asexual seed formation.

Diversity, phylogeny and evolution of the rapidly evolving genus Psidium L. (Myrtaceae, Myrteae).

BACKGROUND AND AIMS: Psidium is the fourthth largest genus of Myrtaceae in the Neotropics. Psidium guajava is widely cultivated in the tropics for its edible fruit. It is commercially under threat due to the disease guava decline. Psidium cattleyanum is one of the 100 most invasive organisms in the world. Knowledge of the phylogenetic relationships within Psidium is poor. We aim to provide a review of the biology, morphology and ecology of Psidium, a phylogenetic tree, an infrageneric classification and a list of species. METHODS: Morphological and geographic data were obtained by studying Psidium in herbaria and in the field between 1988 and 2020. Forty-six herbaria were visited personally. A database of approx. 6000 specimens was constructed, and the literature was reviewed. Thirty species (about a third of the species in the genus) were sampled for molecular phylogenetic inference. Two chloroplast (psbA-trnH and ndhF) and two nuclear (external transcribed spacer and internal transcribed spacer) regions were targeted. Phylogenetic trees were constructed using maximum likelihood (ML; RaxML) and Bayesian inference (BI; MrBayes). KEY RESULTS: Psidium is a monophyletic genus with four major clades recognized as sections. Section Psidium (ten species), to which P. guajava belongs, is sister to the rest of the genus; it is widespread across the Neotropics. Section Obversifolia (six species; restricted to the Brazilian Atlantic Forest), which includes P. cattleyanum, is sister to the innermost clade composed of sister sections Apertiflora (31 species; widespread but most diverse in the Brazilian Atlantic Forest) + Mitranthes (26 species; widespread in dry forests and probably diverse in the Caribbean). Characters associated with diversification within Psidium are discussed. CONCLUSIONS: Research on pre-foliation, colleters, leaf anatomy, leaf physiology, staminal development, placentation and germination associated with the anatomy of the opercular plug is desirable. Studies are biased towards sections Psidium and Obversifolia, with other sections poorly known.

Differential Methylation Patterns in Apomictic vs. Sexual Genotypes of the Diplosporous Grass Eragrostis curvula.

DNA methylation is an epigenetic mechanism by which a methyl group is added to a cytosine or an adenine. When located in a gene/regulatory sequence it may repress or de-repress genes, depending on the context and species. Eragrostis curvula is an apomictic grass in which facultative genotypes increases the frequency of sexual pistils triggered by epigenetic mechanisms. The aim of the present study was to look for correlations between the reproductive mode and specific methylated genes or genomic regions. To do so, plants with contrasting reproductive modes were investigated through MCSeEd (Methylation Context Sensitive Enzyme ddRad) showing higher levels of DNA methylation in apomictic genotypes. Moreover, an increased proportion of differentially methylated positions over the regulatory regions were observed, suggesting its possible role in regulation of gene expression. Interestingly, the methylation pathway was also found to be self-regulated since two of the main genes (ROS1 and ROS4), involved in de-methylation, were found differentially methylated between genotypes with different reproductive behavior. Moreover, this work allowed us to detect several genes regulated by methylation that were previously found as differentially expressed in the comparisons between apomictic and sexual genotypes, linking DNA methylation to differences in reproductive mode.

Genomic Selection in Tropical Forage Grasses: Current Status and Future Applications.

The world population is expected to be larger and wealthier over the next few decades and will require more animal products, such as milk and beef. Tropical regions have great potential to meet this growing global demand, where pasturelands play a major role in supporting increased animal production. Better forage is required in consonance with improved sustainability as the planted area should not increase and larger areas cultivated with one or a few forage species should be avoided. Although, conventional tropical forage breeding has successfully released well-adapted and high-yielding cultivars over the last few decades, genetic gains from these programs have been low in view of the growing food demand worldwide. To guarantee their future impact on livestock production, breeding programs should leverage genotyping, phenotyping, and envirotyping strategies to increase genetic gains. Genomic selection (GS) and genome-wide association studies play a primary role in this process, with the advantage of increasing genetic gain due to greater selection accuracy, reduced cycle time, and increased number of individuals that can be evaluated. This strategy provides solutions to bottlenecks faced by conventional breeding methods, including long breeding cycles and difficulties to evaluate complex traits. Initial results from implementing GS in tropical forage grasses (TFGs) are promising with notable improvements over phenotypic selection alone. However, the practical impact of GS in TFG breeding programs remains unclear. The development of appropriately sized training populations is essential for the evaluation and validation of selection markers based on estimated breeding values. Large panels of single-nucleotide polymorphism markers in different tropical forage species are required for multiple application targets at a reduced cost. In this context, this review highlights the current challenges, achievements, availability, and development of genomic resources and statistical methods for the implementation of GS in TFGs. Additionally, the prediction accuracies from recent experiments and the potential to harness diversity from genebanks are discussed. Although, GS in TFGs is still incipient, the advances in genomic tools and statistical models will speed up its implementation in the foreseeable future. All TFG breeding programs should be prepared for these changes.

Clone worth? Genetic diversity in obligate apomictic Miconia albicans (Melastomataceae).

Apomixis is the asexual production of seeds by plants and, in theory, would render low genetic diversity and even clonal lineages. However, recent studies have shown otherwise, although is not always clear where the genetic diversity of obligate apomicts comes from. We evaluated the genetic diversity among sister seedlings of M. albicans, an obligate apomictic species in Cerrado, Neotropical Savannas in Central Brazil. A total of 50 seedlings from five individuals were analysed using ISSR primers. We obtained 107 fragments, all with good resolution, consistently observed and replicable. The percentage of polymorphic loci ranged from 28.04% to 33.64% and Shannon's information index (I) averaged 0.173. The expected heterozygosity (He) averaged 0.117, similar to the observed for populations of M. albicans and other selfed species. Only two seedlings showed the same genotype (possible clones), but most differed at least for five loci. Most of variance was among progenies (62%), but we found that 38% was within progenies. Genetic distances separated the progenies in two groups, and analogous analyses between individuals reconstructed the original progenies clustering. The results confirmed a relatively high genetic diversity among sister seedling of this obligatory apomictic plant and clones were rare. This diversity can be generated during development, probably by restitutional meiosis or other recombination processes. These differences may accumulate into lineages and populations well adapted to heterogenous Cerrado environment.

Differential Epigenetic Marks Are Associated with Apospory Expressivity in Diploid Hybrids of Paspalum rufum.

Apomixis seems to emerge from the deregulation of preexisting genes involved in sexuality by genetic and/or epigenetic mechanisms. The trait is associated with polyploidy, but diploid individuals of Paspalum rufum can form aposporous embryo sacs and develop clonal seeds. Moreover, diploid hybrid families presented a wide apospory expressivity variation. To locate methylation changes associated with apomixis expressivity, we compare relative DNA methylation levels, at CG, CHG, and CHH contexts, between full-sib P. rufum diploid genotypes presenting differential apospory expressivity. The survey was performed using a methylation content-sensitive enzyme ddRAD (MCSeEd) strategy on samples at premeiosis/meiosis and postmeiosis stages. Based on the relative methylation level, principal component analysis and heatmaps, clearly discriminate samples with contrasting apospory expressivity. Differential methylated contigs (DMCs) showed 14% of homology to known transcripts of Paspalum notatum reproductive transcriptome, and almost half of them were also differentially expressed between apomictic and sexual samples. DMCs showed homologies to genes involved in flower growth, development, and apomixis. Moreover, a high proportion of DMCs aligned on genomic regions associated with apomixis in Setaria italica. Several stage-specific differential methylated sequences were identified as associated with apospory expressivity, which could guide future functional gene characterization in relation to apomixis success at diploid and tetraploid levels.

Proceedings of the 7th Series of Seminars on Advances in Apomixis Research.

These proceedings contain the abstracts for the presentations given at the 7th biennial Seminars on Advances in Apomixis Research, held virtually on 2-3 and 9 December 2020. The first day hosted the kick-off meeting of the EU-funded Mechanisms of Apomictic Development (MAD) project, while the remaining days were dedicated to oral presentations and in-depth exchanges on the latest progress in the field of apomixis and plant reproductive biology research.

A study of the heterochronic sense/antisense RNA representation in florets of sexual and apomictic Paspalum notatum.

BACKGROUND: Apomixis, an asexual mode of plant reproduction, is a genetically heritable trait evolutionarily related to sexuality, which enables the fixation of heterozygous genetic combinations through the development of maternal seeds. Recently, reference floral transcriptomes were generated from sexual and apomictic biotypes of Paspalum notatum, one of the most well-known plant models for the study of apomixis. However, the transcriptome dynamics, the occurrence of apomixis vs. sexual expression heterochronicity across consecutive developmental steps and the orientation of transcription (sense/antisense) remain unexplored. RESULTS: We produced 24 Illumina TruSeq®/ Hiseq 1500 sense/antisense floral transcriptome libraries covering four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis) in biological triplicates, from an obligate apomictic and a full sexual genotype. De novo assemblies with Trinity yielded 103,699 and 100,114 transcripts for the apomictic and sexual samples respectively. A global comparative analysis involving reads from all developmental stages revealed 19,352 differentially expressed sense transcripts, of which 13,205 (68%) and 6147 (32%) were up- and down-regulated in apomictic samples with respect to the sexual ones. Interestingly, 100 differentially expressed antisense transcripts were detected, 55 (55%) of them up- and 45 (45%) down-regulated in apomictic libraries. A stage-by-stage comparative analysis showed a higher number of differentially expressed candidates due to heterochronicity discrimination: the highest number of differential sense transcripts was detected at premeiosis (23,651), followed by meiosis (22,830), postmeiosis (19,100), and anthesis (17,962), while the highest number of differential antisense transcripts were detected at anthesis (495), followed by postmeiosis (164), meiosis (120) and premeiosis (115). Members of the AP2, ARF, MYB and WRKY transcription factor families, as well as the auxin, jasmonate and cytokinin plant hormone families appeared broadly deregulated. Moreover, the chronological expression profile of several well-characterized apomixis controllers was examined in detail. CONCLUSIONS: This work provides a quantitative sense/antisense gene expression catalogue covering several subsequent reproductive developmental stages from premeiosis to anthesis for apomictic and sexual P. notatum, with potential to reveal heterochronic expression between reproductive types and discover sense/antisense mediated regulation. We detected a contrasting transcriptional and hormonal control in apomixis and sexuality as well as specific sense/antisense modulation occurring at the onset of parthenogenesis.

Stomatal size, ploidy and polyembryony in Eriotheca Stellate Trichome Species Complex (Bombacoideae - Malvaceae) in the Cerrados of Brazil.

Geographic parthenogenesis, range expansion of apomictic plants after climate changes, has been described for Northern Hemisphere gametophytic apomicts. But similar trends have been observed for sporophytic apomicts of Cerrado, the savannas in Brazil. Eriotheca pubescens is a common Cerrado tree, an agamic complex of either hexaploid/polyembryonic apomicts or tetraploid/monoembryonic sexual individuals. Some populations have been described as a new species, Eriotheca estevesiae, all included in the Eriotheca Stellate Trichome Species Complex (ESTSC). Since breeding systems and ploidy are clearly associated with polyembryony and stomatal size, we used these ancillary features to map the reproductive and ploidy level traits of E. pubescens and E. estevesiae. Leaves and seeds were collected from individuals of 19 populations. Seeds were evaluated for the presence of polyembryony and leaves for stomatal measurements. Eight populations were monoembryonic while another eight were polyembryonic and for other three, the embryonic pattern was not readily verified. E. pubescens polyembryonic and hexaploid populations formed a homogeneous group, but monoembryonic plants were more variable. E. estevesiae populations were monoembryonic with smaller stomata. In contrast, some E. pubescens monoembryonic populations further south presented larger stomata. Despite these outliers, possibly mixed populations, stomatal size and embryonic pattern differed from northern to southern populations. Embryonic pattern and stomatal size indicated that northernmost populations of Eriotheca STSC (E. estevesiae) are diploid and sexual. Southernmost populations, mostly polyembryonic and with large stomata, are hexaploid and apomictic. This is in agreement with geographic parthenogenesis and range expansion of apomictic lineages to southern habitats available after the last glacial maximum.

SERK genes identification and expression analysis during somatic embryogenesis and sporogenesis of sexual and apomictic Brachiaria brizantha (Syn. Urochloa brizantha).

MAIN CONCLUSION: In Brachiaria brizantha BbrizSERK1, BbrizSERK2 and BbrizSERK3 were identified. SERK expression marks somatic embryogenesis, sexual MMC, and sexual and apomictic PMC. BbrizSERK3 might have a regulatory role in reproductive development. Somatic embryogenesis receptor-like kinase (SERK) consists of plasma membrane receptor genes that have been characterized in various species, associated with several aspects of plant development, including reproduction. SERK genes are involved in anther development and in early embryo development in sexual and asexual seed formation. To comprehend the complexity of the SERK genes and their function in Brachiaria reproduction, we performed a homology-based search in a genomic database of a sexual B. brizantha and identified sequences of three SERK genes, BbrizSERK1, BbrizSERK2, and BbrizSERK3. RNASeq data showed equivalent abundance of BbrizSERK1 and BbrizSERK2 transcripts in ovaries at early megasporogenesis of sexuals and apomicts, while BbrizSERK3 transcripts were more abundant in ovaries of sexuals than in apomicts. BbrizSERK3 results in three coding sequences due to alternative splicing, among them Variant 1 results in a protein with all the predicted domains of a SERK. BbrizSERK transcripts were detected in male reproductive tissues of both sexual and apomictic plants, suggesting a role in controlling anther development. BbrizSERK transcripts were detected early in ovule development, in the integuments, and in the megaspore mother cell of the sexual plant, but not in the cells that give rise to apomictic embryo sacs, suggesting a role in female reproductive development of sexuals. This paper provides evidences that SERK genes plays a role in the onset and establishment of somatic embryogenesis and in the reproductive development of B. brizantha and suggests a distinct role of BbrizSERK in apomixis initiation.

Genes Modulating the Increase in Sexuality in the Facultative Diplosporous Grass Eragrostis curvula under Water Stress Conditions.

Eragrostis curvula presents mainly facultative genotypes that reproduce by diplosporous apomixis, retaining a percentage of sexual pistils that increase under drought and other stressful situations, indicating that some regulators activated by stress could be affecting the apomixis/sexual switch. Water stress experiments were performed in order to associate the increase in sexual embryo sacs with the differential expression of genes in a facultative apomictic cultivar using cytoembryology and RNA sequencing. The percentage of sexual embryo sacs increased from 4 to 24% and 501 out of the 201,011 transcripts were differentially expressed (DE) between control and stressed plants. DE transcripts were compared with previous transcriptomes where apomictic and sexual genotypes were contrasted. The results point as candidates to transcripts related to methylation, ubiquitination, hormone and signal transduction pathways, transcription regulation and cell wall biosynthesis, some acting as a general response to stress and some that are specific to the reproductive mode. We suggest that a DNA glycosylase EcROS1-like could be demethylating, thus de-repressing a gene or genes involved in the sexuality pathways. Many of the other DE transcripts could be part of a complex mechanism that regulates apomixis and sexuality in this grass, the ones in the intersection between control/stress and apo/sex being the strongest candidates.